Chemie und Prozesstechnik
Filtern
Dokumenttyp
- Sonstiges (25) (entfernen)
Referierte Publikation
- nein (25)
Schlagworte
- ELISA (6)
- Traceability (4)
- Antibody (3)
- MALDI-TOF-MS (3)
- SARS-CoV-2 (3)
- ABID (2)
- Antibodies (2)
- COVID-19 (2)
- Corona (2)
- Dynamische Lichtstreuung (2)
Organisationseinheit der BAM
- 1 Analytische Chemie; Referenzmaterialien (25) (entfernen)
The primary screening of hybridoma cells is a time-critical and laborious step during the development of monoclonal antibodies. Often critical errors occur in this phase, which supports the notion that the generation of monoclonal antibodies with hybridoma technology is difficult to control and hence a risky venture. We think that it is crucial to improve the screening process to eliminate most of the immanent deficits of the conventional approach. With this new microarray-based procedure, several advances could be achieved: Selectivity for excellent binders, high throughput, reproducible signals, avoidance of misleading avidity (multivalency) effects, and simultaneous performance of competition experiments. The latter can directly be used to select clones of desired cross-reactivity properties. In this paper, a model system with two excellent clones against carbamazepine, two weak clones and blank supernatant has been designed to examine the effectiveness of the new system. The excellent clones could be detected largely independent of the IgG concentration, which is unknown during the clone screening since the determination and subsequent adjustment of the antibody concentration is not possible in most cases. Furthermore, in this approach, the enrichment, isolation, and purification of IgG for characterization is not necessary. Raw cell culture supernatant can be used directly, even when fetal calf serum (FCS) or other complex media had been used. In addition, an improved method for the oriented antibody-immobilization on epoxy-silanized slides is presented. Based on the results of this model system, we conclude that this approach should be preferable to most other protocols leading to many of false positives, causing expensive and lengthy confirmation steps to weed out the poor clones.
In 2019 over 30 000 people were killed or injured by explosions caused by explosives like TNT, PETN, HMX and RDX. Therefore, highly sensitive assays for the detection of TNT are needed. In this study we compared two commercially available TNT antibodies: A1.1.1 and EW75C with a highly optimized indirect competitive ELISA based on a BSA-TNA conjugate. As a result, a precision profile for both antibodies was determined with a LOD of 170 pmol L-1 for the clone A1.1.1 and a LOD of 3,2 nmol L-1 for the clone EW75C. The measurements showed that the clone A1.1.1 is a highly sensitive antibody for the detection of TNT while the clone EW75C does show medium performance at most.
In the cross-reactivity characterization of both antibodies many substances, closely related to the structure of TNT were tested. Both antibodies showed strong cross reactivity with trinitroaniline and trinitrobenzene. For the clone A1.1.1, which is known to originate from immunization of mice with an TNP-glycine-KLH conjugate, this has to be expected. Interestingly the clone EW75C, which was not characterized yet, showed similar behavior. This suggests a TNA-conjugate as immunogen for the EW75C antibody as well. None of both antibodies showed cross-reactions to the high explosives PETN, HMX and RDX. Also, the cross-reactions of nitro musks with the antibodies were investigated. Despite their prohibition, nitro musks are still used in Asia especially and are particularly popular in India. The overall superior clone A1.1.1 showed a significant cross-reactivity to musk ambrette. For practical reasons the influence of musk ambrette on this assay when used in natural environment should be investigated.
In further experiments, the highly sensitive TNT antibody A1.1.1 was digested with papain to obtain monovalent Fab-fragments. Due to its high stability against the digestion, a custom protocol for the IgG1 subclass of mice, to which the clone A1.1.1 belongs, was developed, resulting in a quantitative digestion of the intact antibody to Fab fragments. The success of the digestion was determined with MALDI-TOF-MS and SDS-PAGE. It was shown that this protocol worked for many different antibodies of IgG1 subclass as well.
The trafficking of illegal drugs by criminal networks at borders, harbors, or airports is an increasing issue in public health as these routes ensure the main supply of illegal drugs. The prevention of drug smuggling, including the installation of scanners and other analytical devices to detect ultra-small traces of drugs within a reasonable time frame, remains a challenge. The presented immunosensor is based on a monolithic affinity column with a large excess of immobilized hapten, which traps fluorescently labeled antibodies as long as the analyte cocaine is absent. In the presence of the drug, some binding sites of the antibody will be blocked, which leads to an immediate breakthrough of the labeled protein, detectable by highly sensitive laser-induced fluorescence with the help of a Peltier-cooled complementary metal-oxide-semiconductor (CMOS) camera. Liquid handling is performed with high-precision syringe pumps and microfluidic chip-based mixing devices and flow cells. The biosensor achieved limits of detection of 23 pM (7 ppt) of cocaine with a response time of 90 seconds and a total assay time below 3 minutes. With surface wipe sampling, the biosensor was able to detect 300 pg of cocaine. This immunosensor belongs to the most sensitive and fastest detectors for cocaine and offers near-continuous analyte measurement.
Zellen, als Grundeinheit für Organismen, bilden einen wesentlichen Eckpfeiler für das Leben. Die Untersuchung von einzelnen Zellen liefert wertvolle Einblicke in fundamentale Prozesse des Lebens. So können Anomalien auf der Zelleebene Indikatoren für Krankheiten sein und durch eine Analyse früh erkannt werden. Weiterhin kann durch ein tiefgreifendes Verständnis von Vorgängen in Zellen auch gezielt Forschung zu z.B. neuen Medikamenten betrieben werden. Damit können die Wirksamkeit erhöht und die Nebenwirkungen reduziert werden.
The outbreak of SARS-CoV-2 in December of 2019, led to a worldwide still on-going pandemic. Since then, several so-called waves of SARS-CoV-2 infections, a time period with a high and fast rising number of new infections, have occurred all over the world. Classic surveillance approaches are hardly applicable, and further, non-detected cases cannot be covered by them. Wastewater-based Epidemiology (WBE) was proven to be a reliable tool for the prediction of new SARS-CoV-2 infection waves, due to the discharge of virus particles in fecal shedding of infectious people. Until now, for the monitoring of SARS-CoV-2 in wastewater, Polymerase Chain Reaction (PCR) is used as analytical tool. Even though PCR is a highly sensitive analytical tool, is presents several disadvantages, such as the need for trained personnel, specific technical equipment, as well as a difficult performance. An analytical tool, to which these disadvantaged do not apply, are immunoassays. In this work, a sandwich Enzyme-Linked Immunosorbent Assay (ELISA), with the immobilization of the capture antibodies on the surface of a Microtiter Plate (MTP), as well as a sandwich Magnetic Bead-Based Assay (MBBA), with immobilization of the capture antibodies on the surface of Magnetic Beads (MBs), targeting the SARS-CoV-2 N-protein, were developed and optimized. Both assay formats were performed with a colorimetric and chemiluminescent detection. The developed assay is composed of the two monoclonal antibodies (mAb) AH2 and DE6 - which was biotinylated in the course of the work - which bind to two different epitops of the antigen N-protein. As tracer, Neutravidin-HRP was used, which binds, through interaction of the Neutravidin with the biotin, to the mAb DE6-Biotin. The assay development and optimization procedure included the investigation of the surface saturation with the mAb AH2, the concentration and dilution of the mAb DE6-Biotin and Neutravidin-HRP, the ideal MBs, the ideal coating as well as dilution buffers, and the colorimetric and chemiluminescent substrates. For the developed and fully optimized colorimetric ELISA, a test midpoint x0 of 388 μg/L, for the chemiluminsecent ELISA of 371 μg/L, for the colorimetric MBBA of 251 μg/L and for the chemiluminescent MBBA of 243 μg/L was obtained. Validation of the colorimetric MBBA was done by measurement of three wastewater samples collected at the Wastewater Treatment Plant (WWTP) Potsdam. Whilst no N-protein could be detected in the samples, by spiking of the wastewater samples with certain concentrations of the N-protein, 10- to 18-times lower concentrations could be back-calculated, which can be attributed to matrix-effects of the wastewater sample. Next to the matrix-effects, also several other reason exist, why no N-protein could be determined in the samples. Because of that, further investigation of the handling, and the measurement of the wastewater samples, as well as the improvement of the assay sensitivity through further optimization steps or exchange of the antibodies, is still necessary.
The publicly available document encapsulates the first version of the Catalogue of Services of the future EC4Safenano Centre (CoS 2019).
The CoS 2019 is structured in 12 Service Categories and 27 Service Topics, for each of the 12 categories considered. This architecture configures a 12 x 27 matrix that allows ordering the potential EC4Safenano offer in 324 types of services/groups of services.
Each type of service/group of services is described, in a simple and friendly way, by means of a specific service sheet: the EC4Safenano - Service Data Sheet (EC4-SDS). These EC4-SDSs allow structuring and summarizing the information of each service, providing the customer with a concise view of characteristics of the service and also the contact details with the service provider.
The CoS 2019 deploys a map of services consisting of a set of 100 EC4-SDSs, covering 7 of the 12 Service Categories and 17 of the 27 Service Topics.
The harmonization of services is visualized as a future necessary step in EC4Safenano, in order to strengthen the offer and provide added value to customers with a growing offer of harmonized services in future versions of the CoS.
The information contained in this document is structured in 3 main sections, as follows:
• Catalogue structure. This section describes in short the main characteristics of the CoS 2019.
• Catalogue content. This section represents the core part of the document and encapsulates the set of 100 SDSs displaying the offer proposed by the CoS 2019.
• Online Catalogue. This section describes the resources implemented by EC4Safenano to facilitate the on-line consultation of the CoS 2019 by customers and other interested parties.
Im Vergleich zu anderen Proteinen ist die Identifizierung von Antikörpern anhand ihrer Sequenz zum Beispiel mittels "peptide mass fingerprinting" schwierig. Da die Sequenzinformation eines Antikörpers aufgrund der hypersomatischen Mutation während der Affinitätsreifung nicht im Genom eines Organismus gespeichert ist, kann die Aminosäuresequenz nicht auf einfachem Weg der DNA-Sequenzierung gewonnen werden. Das ist nur in seltenen Fällen möglich, wenn dem Endanwender der Zellklon der Antikörper-produzierenden Zelle zugänglich ist. Eine Sequenzierung auf Protein-Ebene ist sehr aufwändig und teuer und wird daher fast nie für die Charakterisierung von analytischen Antikörpern verwendet. Der Mangel an Validierung dieser analytischen Antikörper, die bei Experimenten verwendeten werden, löst aber eine Reihe Probleme aus, die die Wiederholbarkeit dieser Experimente schwierig und in einigen Fällen unmöglich macht. Das sorgt jährlich für verschwendete Forschungsgelder in Milliardenhöhe und hindert den wissenschaftlichen Fortschritt.
Ziel der vorliegenden Arbeit war die Entwicklung einer einfachen und schnellen Methode, die es trotzdem ermöglicht, die Identifikation von Antikörpern sicherzustellen. Dazu wurde eine Methode basierend auf dem "peptide mass fingerprinting" gewählt. Das Problem der unbekannten Aminosäuresequenz der Antikörper wurde gelöst, indem lediglich die Peptidmuster der entstehenden Fingerprint-Spektren zur Identifikation herangezogen wurden. MALDI wurde dabei als Ionisationsmethode für die Massenspektrometrie gewählt, da die resultierenden Spektren im Gegensatz zu ESI-MS einfach auszuwerten sind. Auch kann auf eine vorige Trennung der Peptide mittels LC verzichtet werden, was zusätzlich Analysenzeit spart. Für die Proteinspaltung wurde eine simple saure Hydrolyse mittels Ameisensäure gewählt. Im Vergleich zum herkömmlichen Trypsin-Verdau konnten auf zeitraubende Arbeitsschritte wie Denaturierung, Reduktion und Alkylierung der Antikörper verzichtet werden. Die Hydrolyse mittels Ameisensäure wurde bisher nur auf kleine und mittelgroße Proteine angewendet, sodass im ersten Teil dieser Arbeit mehrere Schritte optimiert wurden bevor zufriedenstellende Fingerprint-Spektren von Antikörpern erhalten wurden.
Mykotoxine sind sekundäre Stoffwechselprodukte, die von Schimmelpilzen gebildet werden. Sie sind unvermeidliche Lebensmittelkontaminanten und stellen daher ein ernstes Gesundheitsrisiko für Mensch und Tier dar. Aus diesem Grund hat die Europäische Kommission Vorschriften erlassen, die Höchstwerte für Mykotoxine in bestimmten Lebensmitteln vorsehen. Um die gesetzlichen Grenzwerte einzuhalten, müssen in der Lebensmittelindustrie Mykotoxinanalysen der eingehenden Waren durchgeführt werden.
Gegenwärtig umfasst die Analyse zeitaufwändige Probenvorbereitungsschritte, denen in der Regel eine chromatographische Analyse folgt.
Immunoassays ermöglichen eine Vor-Ort-Analyse, aber in der Regel sind für feste Lebensmittelproben lösungsmittelbasierte Extraktionsschritte erforderlich. Da Immunoassays in der Regel nicht mit den hohen Lösungsmittelkonzentrationen kompatibel sind, die üblicherweise für die Mykotoxinextraktion verwendet werden (z.B. Acetonitril oder Methanol), war es das Ziel dieser Arbeit, ein Extraktionsverfahren für die Mykotoxine Deoxynivalenol und Ochratoxin A aus Weizenmehl und Getreide zu entwickeln, das für Immunoassays und -sensoren verwendet werden kann. Vier verschiedene Lösungsmittel, Methanol (10 %), Ethanol (5 %) und Acetonitril (10 %) sowie Milli-Q-Wasser wurden für die Extraktion des Analyten Deoxynivalenol getestet. Um negative Umweltaspekte zu vermeiden und aufgrund der vergleichbaren Extraktionseffizienz von Milli-Q-Wasser gegenüber den anderen Lösungsmitteln, wurde Milli-Q-Wasser als Extraktionslösung im optimierten Extraktionsprotokoll für Deoxynivalenol verwendet.
Es wurden verschiedene Extraktionsbedingungen, wie die Extraktionszeit, das Lösungsmittel-Feststoff-Verhältnis, der Filtrationsschritt, der Zentrifugationsschritt sowie die Zusammensetzung des Wassers untersucht. Auf der Grundlage der Ergebnisse wurde ein Extraktionsprotokoll festgelegt, mit dem Wiederfindungsraten in einem Bereich von x bis x erzielt wurden. Das Extraktionsprotokoll umfasste die Extraktion von 2 g Weizenmehl mit 40 ml Milli-Q-Wasser für 30 Minuten, die anschließende Zentrifugation bei 3000 U/min für 15 Minuten bei 4 °C und die Filtration des Überstands.